Abstract

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Abstract

No abstract is available for this article.

Abstract

Abstract

A highly flexible enzyme module system (EMS) was developed which allows for the first time the in situ regeneration of deoxythymidine 5′-diphosphate (dTDP)-activated deoxy sugars and furthermore enables us to produce novel sorangiosides in a combinatorial biocatalytic approach using three enzyme modules. The SuSy module with the recombinant plant enzyme sucrose synthase (SuSy) and the deoxy sugar module consisting of the enzymes RmlB (4,6-dehydratase), RmlC (3,5-epimerase) and RmlD (4-ketoreductase) from the biosynthetic pathway of dTDP-β-L-rhamnose were combined with the glycosyltransferase module containing the promiscuous recombinant glycosyltransferase SorF from Sorangium cellulosum So ce12. Kinetic data and the catalytic efficiency were determined for the donor substrates of SorF: dTDP-α-D-glucose, dTDP-β-L-rhamnose, uridine diphosphate (UDP)-α-D-glucose (Glc), and dTDP-6-deoxy-4-keto-α-D-glucose. The synthesis of glucosyl-sorangioside with in situ regeneration of dTDP-Glc was accomplished by combination of SuSy and SorF. The potential of the EMS is demonstrated by combining SuSy, RmlB, RmlC, RmlD with SorF in one-pot for the in situ regeneration of dTDP-activated (deoxy) sugars. The HPLC/MS analysis revealed the formation of rhamnosyl-sorangioside and glucosyl-sorangioside, demonstrating the in situ regeneration of dTDP-β-L-rhamnose and dTDP-α-D-glucose and a cycle number for dTDP higher than 9. Furthermore, NADH (reduced form of nicotinamdie adenine dinucleotide) regeneration with formate dehydrogenase in the reduction step catalyzed by the 4-ketoreductase RmlD could be integrated in the one-pot synthesis yielding similar conversion rates and cycle numbers. In summary, we have established the first in situ regeneration cycle for dTDP-activated (deoxy) sugars by a highly flexible EMS which allows simple exchange of enzymes in the deoxy sugar module and exchange of glycosyltransferases as well as aglycones in the glycosyltransferase module to synthesize new hybrid glycosylated natural products in one-pot.

Myxalamids are potent inhibitors of the eukaryotic electron transport chain produced by different myxobacteria. Here, we describe the identification of the myxalamid biosynthesis gene cluster from Myxococcus xanthus. Additionally, new myxalamids (5–13) have been obtained by mutasynthesis from bkd mutants of M. xanthus and Stigmatella aurantiaca. Moreover, as these bkd mutants are still able to produce myxalamid B (2), the origin of the isobutyryl-CoA (IB-CoA) starter unit required for its biosynthesis has been determined. In a M. xanthus bkd mutant, IB-CoA originates from valine, but in S. aurantiaca this starter unit is derived from α-oxidation of iso-odd fatty acids, thereby connecting primary and secondary metabolism.

Glycosylations are well-established steps in numerous biosynthetic pathways, and the attached sugar moieties often influence the specificity or pharmacology of the modified compounds. The sorangicins belong to the polyketide family of natural products, and exhibit antibiotic activity through inhibition of bacterial RNA polymerase. We have identified the sorangicin biosynthetic gene cluster in the producing myxobacterium Sorangium cellulosum So ce12. Within the cluster, sorF encodes a putative glycosyltransferase. To determine its function in sorangicin biosynthesis, SorF was heterologously expressed as a fusion protein in Escherichia coli. After purification by affinity chromatography, SorF was found to glucosylate sorangicin A in vitro, utilizing UDP-α-D-glucose as the natural donor substrate. Additionally, SorF showed high flexibility towards further UDP- and dTDP-sugars and was able to transfer several other sugar moieties—α-D-galactose, α-D-xylose, β-L-rhamnose, and 6-deoxy-4-keto-α-D-glucose—onto the aglycon. SorF is therefore one of the rare glycosyltransferases able to transfer both D- and L-sugars, and could thus be used to generate novel sorangiosides.

The macrocyclic polyketide kendomycin exhibits antiosteoporotic and antibacterial activity, as well as strong cytotoxicity against multiple human tumor cell lines. Despite the promise of this compound in several therapeutic areas, the cellular target(s) of kendomycin have not been identified to date. We have used a number of approaches, including microscopy, proteomics, and bioinformatics, to investigate the mode of action of kendomycin in mammalian cell cultures. In response to kendomycin treatment, human U-937 tumor cells exhibit depolarization of the mitochondrial membrane, caspase 3 activation, and DNA laddering, consistent with induction of the intrinsic apoptotic pathway. To elucidate possible apoptotic triggers, DIGE and MALDI-TOF were used to identify proteins that are differently regulated in U-937 cells relative to controls. Statistical analysis of the proteomics data by the new web-based application GeneTrail highlighted several significant changes in protein expression, most notably among proteasomal regulatory subunits. Overall, the profile of altered expression closely matches that observed with other tumor cell lines in response to proteasome inhibition. Direct assay in vitro further shows that kendomycin inhibits the chymotrypsin-like activity of the rabbit reticulocyte proteasome, with comparable efficacy to the established inhibitor MG-132. We have also demonstrated that ubiquitinylated proteins accumulate in kendomycin-treated U-937 cells, while vacuolization of the endoplasmic reticulum and mitochondrial swelling are induced in a second cell line derived from kangaroo rat epithelial (PtK₂) cells, phenotypes classically associated with inhibition of the proteasome. This study therefore provides evidence that kendomycin mediates its cytotoxic effects, at least in part, through proteasome inhibition.

It has been proposed that two acyl carrier proteins (ACPs)—TaB and TaE—and two 3-hydroxy-3-methylglutaryl synthases (HMGSs)—TaC and TaF—could constitute two functional ACP-HMGS pairs (TaB/TaC and TaE/TaF) responsible for the incorporation of acetate and propionate units into the myxovirescin A scaffold, leading to the formation of β-methyl and β-ethyl groups, respectively. It has been suggested that three more proteins—TaX and TaY, which are members of the superfamily of enoyl-CoA hydratases (ECHs), and a variant ketosynthase (KS) TaK—are shared between two ACP-HMGS pairs, to give the complete set of enzymes required to perform the β-alkylations. The β-methyl branch is presumably further hydroxylated (by TaH) and methylated to produce the methoxymethyl group observed in myxovirescin A. To substantiate this hypothesis, a series of gene-deletion mutants were created, and the effects of these mutations on myxovirescin production were examined. As predicted, ΔtaB and ΔtaE ACP mutants revealed similar phenotypes to their associated HMGS mutants ΔtaC and ΔtaF, respectively, thus providing direct evidence for the role of TaE/TaF in the formation of the β-ethyl branch and implying a role for TaB/TaC in the formation of the β-methyl group. Production of myxovirescin A was dramatically reduced in a ΔtaK mutant and abolished in both the ΔtaX and the ΔtaY mutant backgrounds. Analysis of a ΔtaH mutant confirmed the role of the cytochrome P450 TaH in hydroxylation of the β-methyl group. Taken together, these experiments support a model in which the discrete ACPs TaB and TaE are compatible only with their associated HMGSs TaC and TaF, respectively, and function in a substrate-specific manner. Both TaB and TaC are essential for myxovirescin production, and the TaB/TaC pair can rescue antibiotic production in the absence of either TaE or TaF. Finally, the reduced level of myxovirescin production in the ΔtaE mutant, relative to the ΔtaF strain, suggests an additional function of the TaE ACP.

Austausch im Eintopf: Die kürzlich beschriebene Reversibilität des Zuckertransfers durch Glycosyltransferasen ermöglicht den Austausch von Zuckern oder Aglyconen und damit die Synthese neuer glycosylierter Substanzen in Eintopfreaktionen (NDP=Nucleosiddiphosphat, GT=Glycosyltransferase).

Unter der Lupe: Der Biosyntheseweg zur Bildung der Aurachin-Alkaloide (gezeigt ist Aurachin A) wurde mithilfe molekularbiologischer Methoden, einschließlich der Klonierung und Sequenzierung des Biosynthesegenclusters im Bakterium Stigmatella aurantiaca aufgeklärt. Der Gencluster enthält eine Typ-II-Polyketidsynthase, die in dieser Form erstmals in einem Gram-negativen Bakterium gefunden wurde.

The cover picture shows the effects of the polyketide kendomycin on the model mammalian cell lines U-937 and PtK₂. Kendomycin induces a multiplicity of cellular responses, including up-regulation of stress-response proteins, vacuolization of the endoplasmic reticulum, accumulation of ubiquitinylated proteins and apoptosis. These effects are consistent with inhibition of the proteasome, an activity that kendomycin also exhibits in vitro. Kendomycin thus represents a new structural type of proteasome inhibitor, with potential utility both in chemical genetics and therapy. Further details can be found in the article by R. Müller et al. on p. 1261 ff.

A bit of give and take: The recently described reversibility of sugar transfer by glycosyltransferases allows the exchange of sugar or aglycon moieties resulting in the formation of novel glycosylated compounds in a one-pot reaction (see picture; NDP=nucleoside diphosphate, GT=glycosyltransferase).

A closer look: The path for the biosynthesis of aurachin alkaloids (aurachin A is shown) has been deduced by molecular biological methods, including the cloning and sequencing of the biosynthetic gene cluster in the bacterium Stigmatella aurantiaca. The gene cluster encodes a type II polyketide synthase, which was discovered in this form for the first time in a Gram-negative bacterium.

Myxococcus xanthus DK1622 is shown to be a producer of myxovirescin (antibiotic TA) antibiotics. The myxovirescin biosynthetic gene cluster spans at least 21 open reading frames (ORFs) and covers a chromosomal region of approximately 83 kb. In silico analysis of myxovirescin ORFs in conjunction with genetic studies suggests the involvement of four type I polyketide synthases (PKSs; TaI, TaL, TaO, and TaP), one major hybrid PKS/NRPS (Ta-1), and a number of monofunctional enzymes similar to the ones involved in type II fatty-acid biosyntesis (FAB). Whereas deletion of either taI or taL causes a dramatic drop in myxovirescin production, deletion of both genes (ΔtaIL) leads to the complete loss of myxovirescin production. These results suggest that both TaI and TaL PKSs might act in conjunction with a methyltransferase, reductases, and a monooxygenase to produce the 2-hydroxyvaleryl–S–ACP starter that is proposed to act as the biosynthetic primer in the initial condensation reaction with glycine. Polymerization of the remaining 11 acetates required for lactone formation is directed by 12 modules of Ta-1, TaO, and TaP megasynthetases. All modules, except for the first module of TaL, lack cognate acyltransferase (AT) domains. Furthermore, deletion of a discrete tandem AT—encoded by taV—blocks myxovirescin production; this suggests an “in trans” mode of action. To embellish the macrocycle with methyl and ethyl moieties, assembly of the myxovirescin scaffold is proposed to switch twice from PKS to 3-hydroxy-3-methylglutaryl–CoA (HMG–CoA)-like biochemistry during biosynthesis. Disruption of the S-adenosylmethionine (SAM)-dependent methyltransferase, TaQ, shifts production toward two novel myxovirescin analogues, designated myxovirescin Q_a and myxovirescin Q_c. NMR analysis of purified myxovirescin Q_a revealed the loss of the methoxy carbon atom. This novel analogue lacks bioactivity against E. coli.

Secondary metabolism involves a broad diversity of biochemical reactions that result in a wide variety of biologically active compounds. Terminal amide formation during the biosynthesis of the myxobacterial electron-transport inhibitor, myxothiazol, was analyzed by heterologous expression of the unique nonribosomal-peptide synthetase, MtaG, and incubation with a synthesized substrate mimic. These experiments provide evidence that the terminal amide is formed from a carrier protein-bound myxothiazol acid that is thioesterified to MtaF. This intermediate is transformed to an amide by extension with glycine and subsequent oxidative cleavage by MtaG. The final steps of melithiazol assembly involve a highly similar protein-bound intermediate (attached to MelF, a homologue of MtaF), which is transformed to an amide by MelG (homologue of MtaG). In this study, we also show that the amide moiety of myxothiazol A can be hydrolyzed in vivo to the formerly unknown free myxothiazol acid by heterologous expression of melJ in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The methyltransferase MelK can finally methylate the acid to give rise to the methyl ester, which is produced as the final product in the melithiazol A biosynthetic pathway. These experiments clarify the role of MelJ and MelK during melithiazol assembly.

Eine molekulare Ursache der Naturstoffvariation. Ein Vergleich der nichtribosomalen Peptidsynthetasen (NRPS) aus den Biosynthesewegen zweier strukturell ähnlicher myxobakterieller Lipopeptide ergab eine Korrelation zwischen dem Überspringen eines Moduls und Punktmutationen mit den Strukturvariationen dieser Naturstoffe. Das Überspringen eines kompletten Moduls während der Myxochromid-S-Biosynthese ist das erste Beispiel für Modul-„Skipping“ in einem NRPS-System (siehe Bild).

“Totgesagte leben länger!” Dieses Sprichwort gilt auch für die Naturstoff-Forschung. Nachdem Naturstoffe als Leitverbindungen durch die Einführung kombinatorischer Synthesetechniken in den Hintergrund gedrängt worden waren, haben sie ihren Rang in der pharmazeutischen Forschung zurückerlangt. Durch Entwicklungen auf diesem Gebiet, das moderne Genomik mit angewandter Biologie und Chemie kombiniert, können Herausforderungen wie das vermehrte Auftreten multiresistenter Bakterien bewältigt werden, da neue Arzneistoffe und Leitstrukturen identifiziert, produziert und in ihrer Struktur verändert werden können. Der große Anteil der Sekundärstoffe und ihrer Derivate unter den klinisch eingesetzten Substanzen erklärt sich dadurch, dass sich Naturstoffe häufiger durch biologische Aktivitäten auszeichnen als Synthetika. Dieser Aufsatz beschreibt den Einfluss der mikrobiellen Genomik auf die Naturstoff-Forschung, insbesondere auf die Suche nach neuen Leitstrukturen und deren Optimierung, zeigt ihre Grenzen auf und gibt Einblicke in mögliche zukünftige Entwicklungen.

The myxobacterium Stigmatella aurantiaca DW4/3–1 harbours an astonishing variety of secondary metabolic gene clusters, at least two of which were found by gene inactivation experiments to be connected to the biosynthesis of previously unknown metabolites. In this study, we elucidate the structures of myxochromides S_1–3, novel cyclic pentapeptide natural products possessing unsaturated polyketide side chains, and identify the corresponding biosynthetic gene locus, made up of six nonribosomal peptide synthetase modules. By analyzing the deduced substrate specificities of the adenylation domains, it is shown that module 4 is most probably skipped during the biosynthetic process. The polyketide synthase MchA harbours only one module and is presumably responsible for the formation of the variable complete polyketide side chains. These data indicate that MchA is responsible for an unusual iterative polyketide chain assembly.

The myxochelins are catecholate-type siderophores produced by a number of myxobacterial strains, and their corresponding biosynthetic gene clusters have been identified in Stigmatella aurantiaca Sg a15,1 and Sorangium cellulosum So ce56; the latter being presented in this work. Biochemical and genetic studies described here further clarify myxochelin biosynthesis. In addition to the myxochelin A biosynthetic complex, the aminotransferase MxcL is required in order to form myxochelin B, starting from 2,3-dihydroxy benzoic acid and L-lysine. Additionally, the substrate specificity of the myxochelin A biosynthetic complex was analyzed in vitro; this led to the formation of novel myxochelin derivatives. Furthermore, MxcD was over-expressed and its function as an active isochorismic acid synthase in Escherichia coli was verified by complementation studies, as was activity in vitro. The organization of the myxochelin gene cluster of S. cellulosum So ce56 was compared to that of the Sg a15 gene cluster. The comparison revealed that although the organization of the biosynthetic genes is completely different, the biosynthesis is most probably extremely similar.

A biosynthetic shunt pathway branching from the mevalonate pathway and providing starter units for branched-chain fatty acid and secondary metabolite biosynthesis has been identified in strains of the myxobacterium Stigmatella aurantiaca. This pathway is upregulated when the branched-chain α-keto acid dehydrogenase gene (bkd) is inactivated, thus impairing the normal branched-chain amino acid degradation process. We previously proposed that, in this pathway, isovaleryl-CoA is derived from 3,3-dimethylacrylyl-CoA (DMA-CoA). Here we show that DMA-CoA is an isomerization product of 3-methylbut-3-enoyl-CoA (3MB-CoA). This compound is directly derived from 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) by a decarboxylation/ dehydration reaction resembling the conversion of mevalonate 5-diphosphate to isopentenyl diphosphate. Incubation of cell-free extracts of a bkd mutant with HMG-CoA gave product(s) with the molecular mass of 3MB-CoA or DMA-CoA. The shunt pathway most likely also operates reversibly and provides an alternative source for the monomers of isoprenoid biosynthesis in myxobacteria that utilize L-leucine as precursor.

Myxobacteria show a high potential for the production of natural compounds that exhibit a wide variety of antibiotic, antifungal, and cytotoxic activities.1, 2 The genus Sorangium is of special biotechnological interest because it produces almost half of the secondary metabolites isolated from these microorganisms. We describe a transposon-mutagenesis approach to identifying the disorazol biosynthetic gene cluster in Sorangium cellulosum So ce12, a producer of multiple natural products. In addition to the highly effective disorazol-type tubulin destabilizers,3–5 S. cellulosum So ce12 produces sorangicins, potent eubacterial RNA polymerase inhibitors,6 bactericidal sorangiolides, and the antifungal chivosazoles.7, 8 To obtain a transposon library of sufficient size suitable for the identification of the presumed biosynthetic gene clusters, an efficient transformation method was developed. We present here the first electroporation protocol for a strain of the genus Sorangium. The transposon library was screened for disorazol-negative mutants. This approach led to the identification of the corresponding trans-acyltransferase core biosynthetic gene cluster together with a region in the chromosome that is likely to be involved in disorazol biosynthesis. A third region in the genome harbors another gene that is presumed to be involved in the regulation of disorazol production. A detailed analysis of the biosynthetic and regulatory genes is presented in this paper.

Klassische Fütterungsexperimente belegen die Beteiligung von nichtribosomaler Peptidsynthese, Polyketidsynthese und Isoprenoidbiosynthese an der Bildung des myxobakteriellen Antibiotikums Leupyrrin (1). Die so erzeugten Strukturelemente bilden nach zusätzlichen Modifizierungen (z. B. durch Semipinakol-ähnliche Umlagerung oder oxidative Bildung einer Methylenbrücke) die einzigartige Struktur von Leupyrrin.

Classical feeding experiments indicate involvement of nonribosomal peptide, polyketide, and isoprenoid biosynthesis in the formation of the myxobacterial antibiotic leupyrrin (1). The resulting structural elements are modified further (e.g. by a semipinacol-like rearrangement and oxidative formation of a methylene bridge) to yield the unique leupyrrin framework.